CpG ODN is a type of oligodeoxynucleotides centered with non-methylated cytosine and guanine nucleotide (CpG). Typically, the CpG is flanked by base sequences in the following manner: 5′PurPurCGPyrPyr 3′, i.e., 2 purines at its 5's end and 2 pyrimidines at its 3′ end. (G Mutwiri, R. Pontarollo, S. BaBIUK. Biological activity of immunostimulatory CpG ODN motif in domestic animals. Veterinary Immunopathology, 2003, 91: 89-103). Studies indicate CpG ODN can activate a variety of immune effector cells, in which non-methylated CpG dinucleotides is believed to be of importance to the immunological activity of the CpG ODN. The DNAs of bacteria, viruses, and invertebrates possess immunological activation function because they have non-methylated CPG ODN sequences. The DNAs of vertebrates, in contrast, do not possess the function because their CpGs are methylated. Immune response against exogenous DNA is elicited by the immune system of the body through the recognition of the unmethylated CpG (Yi A K, Klinman D M, Martin T L, et al. Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway. Immunol., 1996, 157(12):5394-402).
Rabies, also referred to as hydrophobia, takes place in over 60 countries all over the world. (Wolfgang Haupt. Rabies—risk of exposure and current trends in prevention of human cases. Vaccine 1999 (17): 1742-1749). The incidence of the disease in Southeast Asia countries is higher than in other regions of the world. In mainland China, the incidence is between 0.4/100,000-1.58/100,000. Recently, the incidence of rabies grew up rapidly in many countries including China (David W Dreesen. A global review of rabies vaccines for human use. Vaccine, 1997, 15, suppl s2-s6. D. Zienius, J. Bagdonas, A. Dranseika. Epidemiological situation of rabies in Lithuania from 1990 to 2000. Veterinary Microbiology, 2003(93):91-100.), with a death rate of nearly 100%.
Generally, human is infected by rabies virus through for instance, animal such as dog bite, where the animal carries rabies virus. In China, 80%-90% human rabies cases are caused by infected dogs. Human rabies is an acute infectious disease characterized by the invasion of central nervous system (Alan C. Jackson, William H. Wunner. Detection of Rabies Virus Genomic RNA and mRNA in Mouse and Human Brains by Using In Situ Hybridization. Journal of Virology, 1991, 65(6):2839-2844.), the clinical manifestations of which include hydrophobia, anxiety, fear of wind, pharynx spasm, progressing paralysis, etc.
Rabies virus belongs to Rhabdoviridae family, with a size of about 75×180 nm. It is a single minus strand RNA encapsulated by protein capsid, the surface of which is covered by a lipoprotein envelope. The envelope further contains glycoprotein spikes. Rabies virus has immunogenicity, which can not only induce neutral antibodies but also cause RBC aggregation in animals such as chicken and goose. Rabies virus can be simply inactivated by UV radiation, quarternary ammonium compounds, iodine, potassium permanganate, alcohol, formaldehyde, etc. Heating at 100° C. for 2 minutes can also inactivate the virus. Rabies virus is tolerant to low temperatures. It can survive under −70° C. or −4° C. (in lyophilized form) for years.
Currently, there is no effective treatment for rabies infections. Up to now, rabies virus vaccine inoculation (hereafter referred to as rabies vaccine) and anti-rabies serum administration remain the major methods for preventing rabies infections. Presently in China, hamster renal cell vaccine is used as rabies vaccine. The subjects are administered 5 times intramuscularly by injection in a whole course, wherein each administration is taken on days 0, 3, 7, 14 and 30. For severe patients, they are administered 10 times in the whole course, i.e., one administration per day from the date of being bitten to 5 days after the bitten, and the rest of the administrations being taken on days 10, 14, 30 and 90. For people bitten by infected dogs, the average incidence rate is 15%-20%. In comparison, the incidence rate drops to 0.15% after taken a whole course administration.
Hepatitis B is a liver disease caused by hepatitis B virus (HBV), which has severely affected the health of people around the globe. By 2004, the number of HBV patient has reached 400,000,000 worldwide (Lin K W, Kirchner J T. Hepatitis B. Am Fam Physician, 2004 Jan. 1, 69(1):75-82.), most of them are Asians. In China, HBV carriers account for about 10% of the entire population. Presently, the major method to prevent HBV propagation is by way of inoculating HbsAg genetic engineering vaccine, where Aluminum is used as an adjuvant. According to WTO reports, 1 billion dosages of HBV vaccine have been consumed since 1982, which take an important role in combating the disease. The mechanism of the vaccine is that it induces the body to produce/secrete protective antibody IgG1. Although the antibody can neutralize the viruses outside of the cells, it could not thoroughly eliminate latent HBVs inside the infected cells. Furthermore, 10% of the population are low responsive or even have no response to the vaccine. Therefore, what is needed now is to improve the immunocompetence of the present HBV vaccines or to develop new vaccines which can effectively eliminate the latent HBV inside the infected cells. A number of studies on the development of novel HBV genetic engineering vaccines have been carried out from different perspectives by researchers all over the world. Among them, an important topic relates to the finding of an effective adjuvant of HBV vaccine. In recent years, CpG ODN became one of the newly discovered immunological adjuvants proved to be effective.
The result of a variety of experimental studies indicates that CpG ODN can work with HBV vaccine synergistically to induce the production of specific antibodies and elicit CTL response in murine, human, and other primates (Weeranta R D, McCluskie M J, Xu Y. et al. CpG ODN is a novel non-toxic adjuvant which induces stronger immune responses than many conventional adjuvants. Vaccine, 2000, 18: 1755-62.). CpG ODN is considered as a safe and effective adjuvant for HBV vaccine.
In 1988, Davis et al. immunized BALB/c mice with HBsAg and CpG ODN1826 (as adjuvant). The results of HBsAb assay indicated that the HBsAbs produced by the co-administration of HBsAg and CpG ODN as adjuvant is 5 times higher than those produced by the co-administration of HBsAg and aluminum; the HBsAbs produced by the co-administration of HBsAg, and CpG ODN as well as aluminum as adjuvants is 35 times higher than those produced by the co-administration of HBsAg and aluminum; and in control group where only HBsAg was added, no HBsAb or very low lever HBsAb was observed. All these data shows as HBsAg adjuvants, CpG ODN functions better than aluminum in inducing HBsAb production, and CpG ODN further functions synergistically with aluminum. The results of ELISA and 51Cr killing test indicated the combination use of CpG ODN and HBsAg, or the combination use of CpG ODN, aluminum and HbsAg can elicit Th1 immune response in mice, resulting in the production of IgG2a HBsAb and accompanied by HBV-specific CTL response; while the combination use of aluminum and HBsAg mainly elicited Th2 immune response in mice, resulting in the production of IgG1 HBsAb and not accompanied by HBV-specific CTL response. The in vitro antibody staining result of cell surface molecule revealed that the mechanism CpG ODN used to enhance the immunological effectiveness of HBsAg is closely associated with mechanisms through which CpG induces APC to express co-stimulative molecules, and synergistically induces the class switch of the antibodies produced by B lymphocyte (Hartmann Q Weeratna R D, Ballas Z K, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. Immunol, 2000, 164:1617-24). All the above-discussed results suggest CpG ODN is a promising adjuvant for HBV vaccine.